Urine DNA Kit for monitoring HCC recurrence This SBIR phase I application is to test the feasibility of developing HCC urine DNA biomarkers to address the critical unmet need for a noninvasive, sensitive monitoring tool for early detection of hepatocellular carcinoma (HCC) recurrence. HCC incidence has doubled overall in the past two decades, making it one of the fastest growing malignancies in the United States and a leading cause of cancer-related deaths globally. There is greater than a 70% chance of recurrence (~50% of which occurs within 2 years) in HCC patients who undergo curative therapy (resection, transplantation, or local ablation), thus limiting overall survival. Currently, post treatment monitoring guidelines are unclear and patients are typically monitored every 3 months by serum AFP and imaging (such as ultrasound and MRI) for the first 2 years, with increasing intervals thereafter. JBS Science is pioneering the development of assays detecting HCC urine DNA markers for HCC detection and management. This application will apply three already developed and patented short- amplicon assays (TP53 249T, mRASSF1A and mGSTP1), and develop short-amplicon assays for additional known HCC hotspot mutations (CTNNB1 and TERT promoter mutations) for development of a urine DNA kit for monitoring HCC recurrence. This proposal aims to test the feasibility of early detection of HCC recurrence using these five marker assays in patients treated with curative interventions. Two aims are proposed. Aim 1 is for assay development and Aim 2 is to determine the performance of the HCC urine DNA kit for detection of HCC recurrence as compared to serum AFP. We will prospectively collect serial urine samples from at least 120 HCC patients after treatment at follow-up office visits from two clinical sites, Johns Hopkins University and Thomas Jefferson University, for monitoring recurrence. Marker values will be determined for each urine sample by our urine DNA kit and analyzed for the performance of JBS urine DNA kit to screen for HCC recurrence alone or in combination with serum AFP. In addition we will determine whether the urine DNA biomarker test can detect HCC earlier than current imaging (MRI/CT scan). This proposal will lead to the development of a method that can detect HCC recurrence earlier than the current methods by either replacing or combining AFP with a simple and non-invasive urine DNA test. This will bring more patients to sophisticated imaging diagnosis (such as MRI or CT), in a quick and accurate manner in locating the tumor for treatment. 1